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P35-sensitive caspases, MAP kinases and Rho modulate ß-adrenergic induction of apoptosis in mollusc immune cells

Station Biologique de Roscoff, CNRS, Université Paris VI, INSU Place Georges Teissier, B.P. 74, F-29682 Roscoff cedex, France

View larger version (73K): [in a new window]   Fig. 1. Exposure of oyster hemocytes to 1 µM NA for 24 hours induces membranal changes that are indicative of cells undergoing apoptosis. (A) A scanning electron micrograph of a control hemocyte maintained in vitro for 24 hours in the absence of NA. The cell tends to spread on the substrate (arrowhead) and deploys several pseudopods (arrows), whereas (B) an oyster hemocyte incubated in the presence of 1 µM NA exhibits visible membrane blebbing (arrow). (C) Confocal microscopy analysis after FITC-AnnexinV-PI staining shows that phosphatidylserine is absent from the external leaflet of the plasma membrane, whereas (D) FITC-Annexin V binds to the membrane of hemocytes incubated in the presence of NA, demonstrating that in addition to membrane blebbing (arrow), externalization of phosphatidylserine, one of the earliest events in apoptosis, is occuring. Cells in (C) and (D) were fixed after FITC-Annexin V staining to allow localization of nuclear DNA by PI. Scale bars: (A,B) 1 µm, (C,D) 2 µm.

View larger version (70K): [in a new window]   Fig. 2. Flow cytometric analysis of FITC-Annexin V-PI stained hemocytes incubated in the presence of 1 µM NA for 0, 6, 12 and 24 hours. Region R1 includes viable cells, which excude PI and are negative for FITC-Annexin V binding. Region R2 includes apoptotic cells, which are FITC-Annexin V positive but impermeable to PI, and region R3 includes non-viable, necrotic or late apoptotic cells, which are positive for FITC-Annexin V staining and for PI uptake. The number of apoptotic cells increases over time and reaches about 16% of the total number of cells after 24 hours of exposure to 1 µM NA. One representative experiment out of three is shown.

View larger version (14K): [in a new window]   Fig. 3. The apoptosis-inducing effect of NA is dose dependent and significant (P<0.01) at concentrations 1 µM. Cells were incubated for 24 hours in the presence of NA. Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the absence of NA.

View larger version (35K): [in a new window]   Fig. 4. (A) A 24 hour exposure to the -adrenoceptor agonist phenylephrine has no significant effect on the percentage of apoptotic hemocytes, whereas (B) a 24 hour exposure to the ß-adrenoceptor agonist isoproterenol mimicks the apoptosis-inducing effect of NA. (C) Addition of the -adrenoceptor antagonist prazosin 30 minutes prior to the addition of 1 µM NA has no significant effect on the percentage of apoptotic cells whereas (D) addition of the ß-adrenoceptor antagonist propranolol blocks the apoptosis-inducing effect of NA. Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the absence of adrenoceptor agonist (A,B) or in the presence of 1 µM NA alone (C,D). Bas (basal level) indicates the percentage of apoptotic cells in samples incubated in the absence of drugs.

View larger version (22K): [in a new window]   Fig. 5. (A) The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) reduces the percentage of apoptotic cells among ISO-treated hemocytes. (B) The protein kinase A inhibitor H-89 but not the protein kinase C inhibitors staurosporine (Stau) and calphostin C (CalC) reduce the percentage of apoptotic cells among ISO-treated hemocytes. These results indicate that adenylate cyclase and protein kinase A are required for the ß-adrenergic induction of apoptosis. Cells were maintained for 24 hours in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the presence of ISO alone.

View larger version (25K): [in a new window]   Fig. 6. Caspase inhibitors reduce the apoptosis-inducing effect of ISO on oyster hemocytes. (A) The pan-caspase inhibitor Z-VAD-FMK reduces the percentage of apoptotic cells among ISO-treated hemocytes, and (B) transfection of oyster hemocytes with a recombinant plasmid containing the baculovirus p35 gene under the transcriptional control of the gastropod hsp70 gene promoter (hsp p35) was able to reduce apoptosis among ISO-treated hemocytes. Control constructs containing the hsp70 gene promoter alone (hsp only) or the p35 gene alone (p35 only) had no significant effect on hemocyte ISO-induced apoptosis. Cells were maintained for 24 hours in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the presence of ISO alone.

View larger version (24K): [in a new window]   Fig. 7. The MAP kinase kinase inhibitor PD098059 (PD) increases the percentage of apoptotic cells among ISO-treated hemocytes, indicating that MAP kinase signaling is involved in mechanisms that protect hemocytes from apoptosis induced by ß-adrenergic signaling. PD098059 alone had no significant effect on the percentage of apoptotic hemocytes. Cells were maintained for 24h in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the presence of ISO alone.

View larger version (20K): [in a new window]   Fig. 8. Rho signaling is involved in mechanisms that protect hemocytes from ISO-induced apoptosis. Transfection of oyster hemocytes with a recombinant plasmid containing the wild-type (hsp rhoWT) or a more active valine-14 mutant (hsp rho V14) Aplysia californica rho gene under the transcriptional control of the gastropod hsp70 gene promoter was able to reduce apoptosis among ISO-treated hemocytes. Control constructs containing the hsp70 gene promoter alone (hsp only) or the rho genes alone (rhoWT only or rhoV14 only) had no significant effect on hemocyte ISO-induced apoptosis. Cells were maintained for 12 hours in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (* for P<0.05, ** for P<0.01) differences from samples incubated in the presence of ISO alone.